NON-HUMAN MAMMALS FOR THE PRODUCTION OF CHIMERIC ANTIBODIES

• US 20160219850A1
• Filed: 04/11/2016
• Published: 08/04/2016
• Est. Priority Date: 09/30/2008
• Status: Active Grant

• Current Assignee: Ablexis LLC
• Original Assignee: Ablexis LLC
• Inventors: GREEN, Larry, SHIZUYA, Hiroaki
• Granted Patent: US 10,575,504 B2
• Time in Patent Office: Days
• US Class Current: 1/1
• CPC Class Codes: A01K 2207/15 Humanized animals
• CPC Class Codes: A01K 2217/052 inducing gain of function
• CPC Class Codes: A01K 2217/072 maintaining or altering function, i.e. knock in
• CPC Class Codes: A01K 2217/15 Animals comprising multiple alterations of the genome, by transgenesis or homologous recombination, e.g. obtained by cross-breeding
• CPC Class Codes: A01K 2227/105 Murine
• CPC Class Codes: A01K 2267/01 Animal expressing industrially exogenous proteins
• CPC Class Codes: A01K 67/0278 Knock-in vertebrates, e.g. humanised vertebrates
• CPC Class Codes: C07K 16/00 Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies antibodies with enzymatic activity, e.g. abzymes C12N9/0002
• CPC Class Codes: C07K 16/283 against Fc-receptors, e.g. CD16, CD32, CD64 CD23 C07K16/2851
• CPC Class Codes: C07K 16/462 Igs containing a variable region (Fv) from one specie and a constant region (Fc) from another
• CPC Class Codes: C07K 2317/14 Specific host cells or culture conditions, e.g. components, pH or temperature
• CPC Class Codes: C07K 2317/21 from primates, e.g. man
• CPC Class Codes: C07K 2317/24 containing regions, domains or residues from different species, e.g. chimeric, humanized or veneered
• CPC Class Codes: C07K 2317/51 Complete heavy chain or Fd fragment, i.e. VH + CH1
• CPC Class Codes: C07K 2317/515 Complete light chain, i.e. VL + CL
• CPC Class Codes: C07K 2317/52 Constant or Fc region; Isotype
• CPC Class Codes: C07K 2317/522 CH1 domain
• CPC Class Codes: C07K 2317/524 CH2 domain
• CPC Class Codes: C07K 2317/526 CH3 domain
• CPC Class Codes: C07K 2317/53 Hinge
• CPC Class Codes: C07K 2317/56 : variable (Fv) region, i.e. VH and/or VL
• CPC Class Codes: C07K 2317/76 : Antagonist effect on antigen, e.g. neutralization or inhibition of binding
• CPC Class Codes: C07K 2317/92 : Affinity (KD), association rate (Ka), dissociation rate (Kd) or EC50 value
• CPC Class Codes: C07K 2317/94 : Stability, e.g. half-life, pH, temperature or enzyme-resistance
• CPC Class Codes: C12N 15/8509 : for producing genetically modified animals, e.g. transgenic

Abstract

The invention provides knock-in non-human cells and mammals having a genome encoding chimeric antibodies and methods of producing knock-in cells and mammals. Certain aspects of the invention include chimeric antibodies, humanized antibodies, pharmaceutical compositions and kits. Certain aspects of the invention also relate to diagnostic and treatment methods using the antibodies of the invention.

Claims

1. A method for obtaining an antibody that binds to a target antigen, which method comprises:

a. immunizing a transgenic mouse with a target antigen, wherein the transgenic mouse has a genome comprising an immunoglobulin (Ig) heavy chain locus comprising a transgene encoding a chimeric Ig heavy chain, the transgene comprising a portion of a mouse Ig heavy chain locus, the portion comprising all or part of constant region genes encoding a chimeric Ig heavy chain, the transgene comprising a DNA fragment comprising in operable linkage from 5′

to 3′

a DNA sequence of a human variable heavy (VH) segment gene operably linked to a constant region gene comprising a human CH1, human C_{upper hinge}, mouse non-human CH2 and mouse non-human CH3 exons, wherein the human CH1 exon is selected from the group consisting of a human Cμ

, a human Cδ

, a human Cγ

1, a human Cγ

2, a human Cγ

4, a human Cα

, and a human Cε

, thereby the transgenic mouse comprises a genome encoding the chimeric Ig heavy chain; and

b. recovering antibodies comprising the chimeric Ig heavy chain that bind to the predetermined antigen.

2. The method according to claim 1, wherein recovering comprises using hybridomas.

3. The method according to claim 2, further comprising isolating a polynucleotide encoding a variable region of the chimeric Ig heavy chain.

4. The method of claim 3, further comprising recombinantly making a variable region polypeptide encoded by the isolated polynucleotide.

5. The method according to claim 4, wherein recombinantly making comprises an antibody display system, a eukaryotic expression system, a prokaryotic expression system, or an in vitro translation system.

6. The method according to claim 3, wherein the isolated polynucleotide is sequenced and a synthetic nucleic acid molecule is produced from the sequence of the isolated polynucleotide.

7. The method according to claim 6, wherein the synthetic nucleic acid molecule comprises the human VH, the human CH1, a human CH2 and a human CH3.

8. The method according to claim 1, wherein recovering comprises selecting B cells that express the antibodies comprising the chimeric Ig heavy chain that bind to the predetermined antigen.

9. The method according to claim 8, further comprising isolating a polynucleotide encoding a variable region of the chimeric Ig heavy chain from the B cells.

10. The method according to claim 9, wherein the isolating comprises single-cell RT-PCR.

11. The method of claim 9, further comprising recombinantly making a variable region polypeptide encoded by the isolated polynucleotide.

12. The method according to claim 11, wherein recombinantly making comprises an antibody display system, a eukaryotic expression system, a prokaryotic expression system, or an in vitro translation system.

13. The method according to claim 10, wherein the isolated polynucleotide is sequenced and a synthetic nucleic acid molecule is produced from the sequence of the isolated polynucleotide.

14. The method according to claim 13, wherein the synthetic nucleic acid molecule comprises the human VH, the human CH1, a human CH2 and a human CH3.

15. The method of claim 1, further comprising isolating a polynucleotide encoding a variable region of the chimeric Ig heavy chain from B cells of the transgenic mouse.

16. The method of claim 15, further comprising recombinantly making a variable region polypeptide encoded by the isolated polynucleotide.

17. The method according to claim 16, wherein recombinantly making comprises an antibody display system, a eukaryotic expression system, a prokaryotic expression system, or an in vitro translation system.

18. The method according to claim 15, wherein the isolated polynucleotide is sequenced and a synthetic nucleic acid molecule is produced from the sequence of the isolated polynucleotide.

19. The method according to claim 18, wherein the synthetic nucleic acid molecule comprises the human VH, the human CH1, a human CH2 and a human CH3.